This translational research program made important discoveries during the current funding cycle. Melanoma cells growing at low pH rely primarily on the monocarboxylic acid transporter (MCT) rather than the NHE1 to regulate intracellular pH, do not demonstrate a Crabtree effect, and upregulate stress proteins HSP27 and 70 that are responsible for enhanced cytoskeletal stability and hyperthermia resistance. In the presence of excess glucose and an inhibitor of respiration melanoma xenografts are acutely acidified by 0.5-0.7 pH unit and pO2 increases. Normal tissues are not affected. It was found that heat activates a checkpoint inhibiting DNA replication through a trans-acting factor, nucleolin, a key component of the nucleolus that regulates the activity of replication protein A (RPA). Nucleolin translocates from the nucleolus to facilitate the interaction with RPA. These findings support the global hypothesis of this program that growth at low pH provides a mechanism unique to tumors by which melanoma can be selectively sensitized by chemical means to pH-dependent therapeutic agents such as hyperthermia. The long-range goal is to sensitize melanoma to thermoradiotherapy by acute acidification from lactic acidosis produced safely by hyperglycemia combined with inhibitors of respiration or lactate transport. Through four interactive projects and two cores this program will: Develop methods to sensitize melanoma xenografts to thermoradiotherapy in vivo (Project 1); Develop NMR techniques to provide non-invasive analysis of pH, 23Nai, glucose metabolism, blood flow and oxygen consumption during acute acidification of heated and unheated melanoma xenografts (Project 2, Subcontract: Univ. of Pennsylvania); Define the relationship between downregulation of heat shock protein synthesis by acute acidification, apoptotic killing during and after hyperthermia, and clonal survival (Project 3); and Elucidate the molecular mechanisms underlying checkpoint activation in cells exposed to heat and acute acidification as a strategy to abrogate heat-induced checkpoint response (Project 4, Subcontract: Univ. of Essen). The measurement of pHe and pHi in vitro and the identification of new methods to inhibit proton transport in melanoma cells grown at low pH will be performed in Core A to benefit all projects. Core B provides biostatistical, electronic communication and data management support for all projects. Results will help direct a Phase I/II clinical trial to demonstrate the effects of acute acidification on melanoma response to hyperthermic limb perfusion, and to identify clinically applicable measures of acidification and bioenergetics to predict response to hyperthermic therapy.